Lyme disease caused by Borrelia burgdorferi, and other tick-borne diseases cause substantial morbidity in the US. Even so, many infections remain undiagnosed. Though more than $500 million is spent annually in the US for about 3.5 million Lyme serological diagnostic tests, available tests lack desired levels of combined sensitivity and specificity and the capability to detect the many distinct tick-transmitted infections that can occur. The aim of this Phase II project is to develop and validate a diagnostic laboratory developed test (LDT) that exhibits improved sensitivity and specificity, and lower cost, when compared to current two-tier testing and enables simultaneous detection of multiple tick-borne infectious organisms. Digital serology is a diagnostic platform that allows for rapid development of new diagnostic tests with improved performance, and combination of an arbitrary number of tests into a single assay without increasing cost. Digital serology uses large random peptide libraries, next- generation sequencing (NGS), and custom bioinformatics tools to simultaneously map the diverse spectrum of immunogenic antigen epitopes targeted for a given disease. The first Aim of this this project is to discover and optimize IgG and IgM epitope motif diagnostic panels highly specific to tick-borne infections including Borellia burgdorferi, Babesia microti, Anaplasma phagocytophilum, and Ehrlichia chaffeensis, and additional Borrelia species B. mayonii and B. miyamotoi. Using well-characterized specimens for each disease, infection- specific peptide motifs will be identified and optimized bioinformatically using motif discovery software and a database of 1000 epitope repertoires from individuals with other diseases and healthy controls. The second Aim of this project is to fully analytically validate panels for at least four different infections using large validation sets. Reproducibility, cross-reactivity, potentially interfering substances, and storage and transport stability will be determined for each test. This project is expected to result in a multiplexed test for tick-borne diseases that is expandable to detect the many distinct infections that can occur from a tick bite and that exhibits improved diagnostic performance and reduced cost.